Mass spectrometry is rapidly becoming a powerful method for the structural elucidation of macromolecular complexes, and can provide complimentary information to conventional biophysical methods. Intact complexes can be lifted out of solution with electrospray ionization, which adds many charges to these molecular assemblies. Experiments have shown that in numerous cases, these gas-phase ions retain many elements of their solution-phase structure. A direct mass measurement provides a rapid measure of the stoichiometry of complexes, even for heterogeneous mixtures. Fragmentation of complexes can yield information about the connectivity of subunits within the complex. Additionally, ion mobility-based approaches are beginning to provide cross-sectional measurements of complexes, making it possible to obtain complementary shape information. A challenge in applying mass spectrometry to even larger complexes is sample heterogeneity, which can result in unresolved charge-state distributions. An additional challenge is that the sensitivity of many mass spectrometers decreases with increasing mass-to-charge ratio (m/z), which makes it more difficult to detect complexes with molecular weights of several mega Daltons and higher with high sensitivity. One aspect of electrospray ionization is that the degree of charging generally increases with molecular size. This makes charge detection mass spectrometry an attractive means of analyzing large macromolecular complexes, because it has the advantage that individual ions possessing multiple charges can be readily detected. Because charge will increase with size, the sensitivity of this technique actually improves with increasing molecular weight. The heterogeneity of the sample does not interfere with mass or mobility measurements, because each ion is analyzed individually. We plan to develop a Single Particle Analyzer of Mass and Mobility (SPAMM), which will incorporate m/z and charge detection measurements so that the masses of individual ions can be rapidly measured. Tandem mass spectrometry capabilities will be incorporated into this dual electrostatic ion trap instrument which will make it possible to measure a fragmentation spectrum of an individual ion of known mass (initial studies will focus on infrared multiple photon dissociation, although other methods could also be incorporated). Finally, ion mobility capabilities will be integrated into this device to make it possible to measure the mass of an ion, and subsequently obtain its absolute collision cross section, which provides information about the shape of the complex. Cross sections can also be obtained for fragments generated by the dissociation of a complex, providing information about the contribution of subunit connectivity to the overall structure of the complex. Because the induced current in this device is directly proportional to the number of charges on the macromolecular complex, sensitivity should increase with increasing particle size making this method applicable too much larger heterogeneous complexes than can be currently analyzed with commercially available mass spectrometers. PUBLIC HEALTH RELEVANCE: Macromolecular complexes play important roles in cellular function, but characterizing the structures of large complexes (tens of mega Daltons and higher) can be challenging owing to heterogeneity that can complicate many conventional methods of analysis. We aim to construct a new instrument that is capable of rapidly measuring the mass, absolute cross section and tandem mass spectra of individual ions of macromolecular complexes at a rate of tens to hundreds of molecules per minute. This would be a high-throuput method to structurally characterize macromolecular complexes, which should aid in understanding their function and make it possible to rapidly screen therapeutic agents that could regulate the activity of macromolecular complexes in a size range not accessible by other mass spectrometry methods.